Stress response mechanisms in bacteria helps them sustain stressful and competitive environments caused by nutrient limitation. Often these stresses give rise adaptive phenotypes that are evolutionarily fitter under nutrient limitation. These phenotypes possess favorable adaptations that help them scavenge and recycle amino acids, carbohydrates, and lipids from other cells, thus playing a crucial role in their survival.
The objective of this project was to develop a systematic procedure to produce evolutionarily fitter populations of E.coli to test the following hypotheses - 1 Bacteria that show increased fitness after being subjected to stress and can outcompete their naive counterparts in a limited resource environment. 2 This increased competitiveness could be exploited to develop a new class of metabolically efficient, contamination resistant host organisms for industrial biotechnology.
{\it E.coli} cultures were exposed to a nutritionally stressful scenario by aging them in fed-batch cultures. Upon competing the aged cultures with the naive ones it was observed that the aged cultures prevail over the naive ones. This is due to a previously observed phenomenon called Growth Advantage in Stationary Phase (GASP), which is believed to be the result of beneficial physiological changes in cells due to imposed nutritional stress.
The metabolic burden imposed on the cells was measured by calculating the doubling time. The doubling time served as a proxy for calculating the fitness of stress hardened cells. Metabolic burden for cells containing plasmids and cells where this plasmid was integrated on the chromosome was measured to characterize the relative metabolic advantage of the integrands over cells with. It was observed that the cells with plasmids experienced higher metabolic burden.
Heterologous protein expression in the stress hardened cells was analyzed to determine if the physiological changes influence protein expression. GFP was used for testing the protein expression. The stress hardened cells showed an approximate two fold increase in the expression of GFP which was a surprising result as the increased production of GFP is not beneficial for the cell in itself.
The stress hardened GASP phenotypes could be used as a chassis for producing commercially relevant proteins and antibiotics. The GASP phenotype is contamination resistant as it is capable of surviving in a nutrient limited condition. This phenotype shows slower growth rate and increased protein production implying that these cells could produce more product for the same amount of materials hence reducing the production cost. Thus, the GASP phenotype could serve a robust chassis for synthetic biology because of the beneficial adaptations it acquires.